Guanidine-HCl (Gu-HCl) or Guanidine Thioisocyanate (GuSCN) can solubilize
both standard and low-melting agarose at 57o C. In the presence
of high salt DNA binds to silica particles then the silica with adsorbed
DNA washed to remove salt and impurities from the original sample, and
the clean DNA eluted in water or TE buffer. This isolation method of DNA
is faster (20 minutes typically) and easier to perform than the other organic-based
extraction method. This method also replaces the Potassium Iodide (KI)
based procedure, where free Iodine may modify the purified DNA. When using
silica adsorption method for isolating DNA from agarose gels, it is important
to note that the use of TBE buffer (Tris-borate-EDTA) can inhibit the ability
of DNA to bind to silica, thus lowering recovery efficiency.
Silica extraction works with a wide size range of DNA and allows efficient
recovery (90%) of product. Using silica you can purify DNA as small as
100bp. There is no upper size limit on recovery efficiency of DNA. However,
precautions should be taken during purification of longer DNA fragments
(20kb) to avoid shearing. Proteins and RNA do not bind to silica powder
and are eliminated during washes and for this reason it is also an ideal
tool to purify and concentrate DNA directly from various reaction mixtures.
Because silica does not bind oligonucleotides with high efficiency (<50
bp), this method can also be used to remove low molecular weight oligonucletides
or nucleotides from DNA. Silica can also be used to remove RNA from DNA,
because RNA does not bind to silica. Primers, unincorporated nucleotides,
the excess of linkers, enzymes and salts, residual phenol, chloroform,
ethidium bromide are separated from the DNA. Therefore, it may be applied
when a single product is generated by PCR, whereas the agarose gel electrophoresis
allows the independent purification of several PCR products from the same
reaction. The purified DNA is suitable for any molecular biology procedures:
restriction digestion, cloning, sequencing, etc. Small amounts of silica
do not inhibit enzymatic reactions, therefore silica bound DNA can be used
for PCR or enzymatic cleavages without prior elution of DNA. The silica
method can be used for the following purposes:

The silica mix (pre-made up to 10 ml) and 6M Gu-HCl or 6M GuSCN solution
can be stored in a refrigerator at +4o C up to six months. The
prepared wash buffer should be stored in the freezer at -20o
C or made up fresh.
If the silica powder suspension looses liquid, add sterile distilled
water to a volume that is approximately equal to the volume of the solid.

Step 1. Run the agarose gel to separate DNA fragments.
Estimate the concentration of DNA by comparing its intensity with that
of a DNA standard of a similar size and known concentration. Cut agarose
gel band containing the desired DNA. (Minimize the time of UV exposure
as much as it is possible.) Determine an approximate volume of gel slice
by weight (1g equals approximately 1ml) and place the slice into a plastic
tube.
Step 2. Add at least 3 volumes of 6M Gu-HCl or 6M GuSCN
solution (the final concentration must be at least 4 M Gu-HCl). Incubate
10 minutes at 57o C to dissolve agarose (mix occasionally to
promote agarose solubilization). If the solubilization is incomplete add
one more volume of 6M Gu-HCl and incubate 5 more minutes.
Step 3. Add the resuspended silica powder suspension.
Up to 2.0 mg of DNA add 5 ml
of silica powder suspension, above 2.0 mg of
DNA add 2 ml of silica powder suspension per
1 mg of DNA and incubate for 5 minutes at 57o
C. (mix occasionally to keep silica resuspended in the solution, do not
vortex)
Step 4. Spin (all centrifugation steps are at 10,000
rpm in a tabletop microcentrifuge) silica powder/DNA complex for 15
seconds to form a pellet . Remove the supernatant. The pelleted silica
powder/DNA is resuspended in 500 m l of 6M Gu-HCl
solution to dissolve any agarose that has not dissolved during step 2.
Place suspension in a 57o C water bath for one minute and pellet
at 10,000 rpm for 15 seconds. Remove the supernatant.
Step 5. Add 500 m l of ice
cold wash buffer to the pellet. Resuspend the pellet in the wash buffer
by pipetting or vortexing the pellet (do not vortex high molecular weight
DNA). Centrifuge for 15 seconds in the centrifuge and discard the
supernatant. Repeat the wash procedure two more times. During each wash
the pellet should be resuspended completely. After the supernatant from
the last wash has been removed, spin the tube again and remove the remaining
liquid with pipette tip. The silica should be relatively ethanol free otherwise
the remaining ethanol may inhibit enzyme reactions (the silica can be airdryed
or vacuum dried for a few minutes, however overdrying my lead to poor recovery).
Step 6. Resuspend the silica powder/ DNA with TE buffer
or water by vortexing/or pipetting. Use at least twice the volume of the
original silica mixture used.Incubate the tube at 570
C for 5-10 minutes. Mix and centrifuge for 30 seconds and carefully remove
the supernatant containing the eluted DNA and place in a new tube. You
can increase the elution efficiency using a large volume and performing
2-3 elution cycles with the new portion of eluant. Typically yields are
greater than 80% in the first elution. In order to remove small amounts
of the silica powder present in the eluate, spin the tube again for 30seconds
in a table-top centrifuge and transfer the supernatant into a new tube
(small amount of silica do not interfere with any type of enzyme assays,
we frequently use silica/DNA complex for PCR, if the amount of silica is
less than 5 m l). Eluted DNA can be used immediately
in enzymatic or other manipulations.
Elute DNA into water or TE buffer.

Recovering DNA
from Polymerase Chain Reactions

Step 1. For recovering PCR products from residual reaction
compounds combine PCR reaction with an equal volume of chloroform (CHCl3)
in the PCR reaction tube to remove the mineral oil. Vortex the tube for
10-15 seconds to mix the layers. Centrifuge the reaction tube for 2 minutes
in a microcentrifuge at 10,000 rpm to separate the aqueous upper and organic
lower layers. Transfer the upper layer, containing DNA to a new microcentrifuge
tube.
Step 2. Measure the volume of PCR product.Add
at least 3 volumes of 6 M Gu-HCl or 6 M GuSCN solution and mix.
Step 3. Add the resuspended silica powder suspension.
Up to 2 mg of DNA add 5 ml
of silica powder suspension, above 2.0 mg of
DNA add 2 ml of silica powder suspension per
1 mg of DNA and incubate for 5 minutes at 57o
C (mix occasionally to keep silica resuspended in the solution).
Step 4. Spin (all centrifugation steps are at 10,000
rpm in a tabletop microcentrifuge) silica powder/DNA complex for 15
seconds to form a pellet. Remove the supernatant.
Step 5. Add 500 ml of ice
cold wash buffer to the pellet. Resuspend the pellet in the wash buffer
by pipetting or vortexing the pellet. Centrifuge for 15 seconds
in the centrifuge and discard the supernatant. Repeat the wash procedure
two more times. During each wash the pellet should be resuspended completely.
After the supernatant from the last wash has been removed, spin the tube
again and remove the remaining liquid with pipette tip. The silica should
be relatively ethanol free otherwise the remaining ethanol may inhibit
enzyme reactions (the silica can be airdryed or vacuum dried for a few
minutes, however overdrying my lead to poor recovery).
Step 6. Resuspend the silica powder/ DNA with TE buffer
or water by vortexing/or pipetting. Use at least twice the volume of the
original silica mixture used.Incubate the tube at 570
C for 5-10 minutes. Mix and centrifuge for 30 seconds and carefully remove
the supernatant containing the eluted DNA and place in a new tube. You
can increase the elution efficiency using a large volume and performing
2-3 elution cycles with the new portion of eluant. Typically yields are
greater than 80% in the first elution. In order to remove small amounts
of the silica powder present in the eluate, spin the tube again for 30seconds
in a table-top centrifuge and transfer the supernatant into a new tube
(small amount of silica do not interfere with any type of enzyme assays,
we frequently use silica/DNA complex for PCR, if the amount of silica is
less than 5 ml). Eluted DNA can be used immediately
in enzymatic or other manipulations.

Troubleshooting guide
of the gel extraction procedure

Possible reason

How to prevent the problem

Low yield of DNA

Agarose gel was not completely solubilized

The incubation temperature for solubilization
should be 57o C.
Insufficient amount of Gu-HCl (it should be at least 3 x the volume
of gel slice).

Insufficient mixing

Mix the DNA/silica mixture every 2-3 minutes.
Sedimented silica do not get contact with the DNA solution

Ethanol concentration is lower than 50% in the
wash.

Be sure that 95% or 100% ethanol is used to
make up the wash solution. Store prepared wash buffer at -20o
C to avoid evaporation.

To much ethanol from the wash buffer remains
in the pellet during elution

Before eluting the DNA completely remove wash
buffer with pipette tip and then if necessary airdry the pellet thoroughly.

Pellet is too dry

Do not vacuum dry the pellet to long.

Enzymatic reactions using recovered DNA do not proceed

Ethanol from the wash buffer remains in the
pellet during DNA elution

Before eluting the DNA, completely remove the
wash buffer with pipette tip and then if necessary airdry the pellet thoroughly.

Isolation
of picogram quantities of DNA from tissue sections

Recovering small amount of DNA from tissues treated
with various chemicals

Step 1. For recovering DNA (less than one microgram)
for PCR from tissues, which has been treated with various chemicals ( such
as paraffin, tissue glue, or cover slip glue), treat the tissue with toluene,
xylene or other organic solvent which solubilize the agent. Generally,
xylene should solubilize the majority of chemicals used in glues or paraffin.
Resuspend the tissue in at least 10-fold excess of xylene. Incubate at
room temperature for 10 minutes with occasional vortexing. Centrifuge at
10,000 rpm for 30 seconds to pellet the tissue. Resuspend the tissue in
fresh Xylene, vortex and centrifuge again. Resuspend the tissue in 95%
ethanol and centrifuge again. Repeat the wash once more. Dry the tissue
in vacuum.
Step 2. Add at least 100 ml
of 6 M Gu-HCl or 6 M GuSCN solution and mix. Incubate at room temperature
(or 60o C if the tissue contains lot of keratin) for 10-15 minutes
to solubilize the proteins and DNA.
Step 3. Add 10 ml of silica
powder suspension, and incubate for 5 minutes at 57o C (mix
occasionally to keep silica resuspended in the solution).
Step 4. Spin (all centrifugation steps are at 10,000
rpm in a tabletop microcentrifuge) silica powder/DNA complex for 15
seconds to form a pellet. Remove the supernatant.
Step 5. Add 500 ml of ice
cold wash buffer to the pellet. Resuspend the pellet in the wash buffer
by vortexing the pellet. Centrifuge for 15 seconds in the centrifuge
and discard the supernatant. Repeat the wash procedure two more times.
During each wash the pellet should be resuspended completely. After the
supernatant from the last wash has been removed, spin the tube again and
remove the remaining liquid with pipette tip. The silica should be relatively
ethanol free otherwise the remaining ethanol may inhibit enzyme reactions
(the silica can be airdryed or vacuum dried for a few minutes, however
overdrying my lead to poor recovery). One way to dry it to add 10
ml
of water to the silica and dry it in vacuum for 10-15 minutes. Ethanol
will evaporate rapidly, but the remaining water will not allow the silica
to dry out.
Step 6. Resuspend the silica powder/ DNA in water by
vortexing in the amount of water that will be used in direct PCR reaction.
Use this mix for PCR.

Alternatively if you try to use the same material for several PCR reaction
the mixture can be divided into several tubes or the DNA can be eluted
with water or TE buffer. Use at least twice the volume of the original
silica mixture used.Incubate the tube at 570
C for 5-10 minutes. Mix and centrifuge for 30 seconds and carefully remove
the supernatant containing the eluted DNA and place in a new tube.